Development and validation of a sensitive quantification method for hematoporphyrin monomethyl ether in plasma using high-performance liquid chromatography with fluorescence detection

A rapid, sensitive, precise and specific method for determination of hematoporphyrin monomethyl ether (HMME), a novel photodynamic therapy (PDT) drug, was developed and validated using high-performance liquid chromatography (HPLC) with fluorescence detection. HMME was isolated from the plasma by a single-step liquid-liquid extraction with ethyl acetate. The analyte and internal standard fluorescein were baseline separated on a Diamonsil C₁₈ analytical column (4.6 × 150 mm, 5 μm) and analyzed using a fluorescence detector with the excitation and emission wavelengths set at 395 and 613 nm, respectively. The method was linear in the concentration range 0.025-5 μg/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL. The inter-and intra-day accuracies and precisions were all within 10% and the mean recoveries of HMME and fluorescein were 95 ± 3.7 and 90 ± 2.3%, respectively. The analyte was stable during all sample storage, preparation and analysis periods. This method was successfully applied to a pharmacokinetic study after a single-dose intravenous administration of HMME (5 mg/kg) to beagle dogs. This method was reproducible and sensitive enough for the pharmacokinetic study of HMME. Based on the results of the pharmacokinetic study, we suggest that a rather long light-avoiding time is essential for patients under HMME therapy.